Forensic DNA- Profiling (Fingerprinting): Using Restriction Enzymes

1. Forensic DNA- Profiling (Fingerprinting): Using Restriction Enzymes

2. DNA ProfilingFingerprinting Real WorldApplications • Crime scene • Human relatedness • Paternity • Animal relatedness • Anthropology studies • Disease-causing organisms • Food identification • Human remains • Monitoring transplants

3. DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus

4. Model of DNADNA is Comprised of Four Base Pairs

5. Deoxyribonucleic Acid (DNA)

6. DNASchematic O Phosphate O P O Base O O CH2 Sugar O O O P Phosphate Base O O CH2 Sugar OH

7. DNA Restriction Enzymes • Evolved by bacteria to protect against viral DNA infection • Endonucleases = cleave within DNA strands • Over 3,000 known enzymes

8. 5 vs 3 Prime Overhang Enzyme cuts • Generates 5 prime overhang

9. Enzyme Site Recognition Restriction site Palindrome • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 2 Fragment 1

10. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

11. DNA FingerprintingProcedureOverview

12. Animations & Videos • Videos: • How restriction enzymes work to create DNA profiles: • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# • How micropipets work: • http://www.youtube.com/watch?v=kCm2t1SO2IU • How electrophoresis works • http://learn.genetics.utah.edu/content/labs/gel/ • PCR: modern method of DNA profiling http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

13. LaboratoryQuick Guide

14. AgaroseElectrophoresisLoading • Electrical current carries negatively-charged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

15. AgaroseElectrophoresisRunning • Agarose gel sieves DNA fragments according to size – Small fragments move farther than large fragments Gel running Power Supply

16. Analysis of Stained Gel Determine restriction fragment sizes • Create standard curve using DNA marker • Measure distance traveled by restriction fragments • Determine size of DNA fragments Identify the related samples

17. DNA DigestionTemperature Why incubate at 37°C? • Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? • Too hot = enzyme may be denatured (killed) • Too cool = enzyme activity lowered, requiring longer digestion time

18. PstI EcoRI Restriction Fragment Length PolymorphismRFLP GAATTC GTTAAC CTGCAG GAGCTC Allele 1 1 2 3 CGGCAG GCGCTC GAATTC GTTAAC Allele 2 3 Fragment 1+2 Different Base Pairs No restriction site M A-1 A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments +

19. Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0